Small molecules targeting the bromodomain-acetyl lysine interaction have emerged as novel epigenetic therapeutics in hematological and virological disease. We showed recently that these inhibitors reactivate HIV from latency in cultured cells and primary T-cell models of latency. Importantly, our data demonstrate that bromodomain inhibitors activate HIV latency by a Tat-independent mechanism, enhance release of active P-TEFb from the inhibitory 7SK ribonucleoprotein complex and implicate an unrecognized bromodomain and extraterminal domain (BET) family member, BRD2, in establishing HIV latency. We propose, in a multi-investigator effort, a set of highly integrated experiments to advance our understanding how the targeting of bromodomain proteins reverses HIV latency at the molecular level with a specific focus on primary latently infected T cells. The proposed studies represent a new line of investigations within the CARE Collaboratory and are designed to conclusively determine if bromodomain inhibitors represent a promising new strategy to purge HIV from latency in patients. In the first specific aim, the laboratories of Melanie Ott, Matija Peterlin, Jonathan Karn and Leor Weinberger propose to characterize the molecular mechanism of bromodomain inhibitor action in HIV latency. Specifically, we will perform an in-depth characterization of the interaction of BRD2 and BRD8 with P-TEFb and associated candidate proteins and will apply 3C to 5C chromosome conformation capture assays as well as single-cell time-lapse microscopy combined with computational modeling to study transcriptional changes induced by bromodomain inhibitors at the HIV LTR. In the second specific aim, the laboratories of Robert Siliciano, David Margolis, Vicente Planelles and Eric Verdin will perform comprehensive studies in primary T cell models and patient-derived cells to test the therapeutic efficiencies of bromodomain inhibitors alone or in combination with other latency-purging drugs. Specific emphasis lies on the knockdown of individual BET proteins in primary T cells and studies of a new dually fluorescence-labeled reporter virus that allows identification of latently infected T cells without prior reactivation. Collectively, these studies will significantly enhance our molecular understanding of the role of bromodomain-containing proteins in HIV transcription and will inform novel drug development strategies to effectively purge viral reservoirs in HIV-infected individuals.